Organelle identity and development rely on a complex set of intracellular protein trafficking systems that mediate the specific targeting of nuclear-encoded proteins to their proper subcellular compartment. Not only must these trafficking systems maintain a high degree of specificity, they must adapt to accommodate dramatic changes in the levels and composition of trafficking substrates imposed by developmental events. Defects in trafficking systems lead to numerous human disorders, including Zellweger syndrome (peroxisome biogenesis) and deafness/dystonia syndrome (mitochondrial protein import), or have lethal consequences that prevent cell growth and development. The study of protein import into plant chloroplasts is a powerful model for investigating protein trafficking and organelle biogenesis in eukaryotic cells because it allows combined biochemical and genetic analyses in a multicellular organism. The long-term goal of this research is to define the molecular mechanism of the import process and understand the role of import in plastid development. This proposal focuses on the role of two GTPases of the translocon at the outer envelope membrane of chloroplasts (Toc complex), Toc34 and Toc159, in preprotein recognition and the regulation of membrane translocation. We will test the hypothesis that Toc159 serves as the initial preprotein receptor and that GTP-regulated heterodimerization with Toc34 serves to regulate transfer of preproteins into the membrane translocation channel, thereby committing preproteins to the import pathway. To this end, we will take advantage of null mutants of Arabidopsis Toc159 (at Toc159) and Toc34 (atToc33) to establish the hierarchy of Toc GTPase function and examine the specific roles of each protein in the import reaction. In addition, combined biochemical and genetic approaches will be used to address the role of the GTPase activities of the Toc components in regulating preprotein recognition and access to the translocon channel. The final aim will test the hypothesis that the different members of the Toc159 and Toc34 families represent distinct pathways for protein targeting to plastids, and that separate pathways have evolved to ensure balanced import of essential proteins during plastid development. The structural basis for the formation and function of distinct translocons will be defined. This information, combined with information on the tissue-specific defects of mutants in each of the four at Toc159 genes, will provide insight into the roles of distinct protein import pathways during organelle and tissue development.